The Role Of Toll Like Receptor 3 In Placenta HBV Infection

OBJECTIVE:The aim of this paper is to explore the role of Toll-Like Receptor 3(TLR3) in term placenta in placenta HBV Infection.By studing in population and in vitro,we explore the expression and distribution of Toll-Like Receptor 3 in term human placenta from normal and HBsAg-positive pregnant women at gene and protein level,the relationship of Toll-Like Receptor 3 in term human placenta and hepatitis B virus(HBV) intrauterine infections.On the basis of the clues from population study,we explore the biological role of TLR3 in HBV infection in placenta by a series of experiments in vitro further more.①To explore the expression and distribution of Toll-Like Receptor 3 in term human placenta from normal and HBsAg-positive pregnant women.To explore the relationship of Toll-Like Receptor 3 in term human placenta with hepatitis B virus(HBV) infection,the relationship between TLR3 mRNA in term human placenta and HBV copies in peripheral blood, the relationship between TLR3 mRNA in term human placenta and HBV intrauterine infections.②To explore the location of TLR3 and HBsAg in placenta,the relationship between TLR3 and placenta HBV infection at the protein level.③To construct a model of HBV infecting the human trophoblast cell line in vitro.To explore the change of TLR3 mRNA expression exposed to HBV in the human trophoblast cell line Bewo,the capacity of TLR3 in Bewo to responses towards HBV infection.METHODS:①Both semi-quantitative RT-PCR and quantitative Real-time RT-PCR were conducted to detect expression of TLR3 mRNA in term human placenta from normal and HBsAg-positive pregnant women.Immunohistochemistry ABC was conducted to detect distribution of TLR3 in term placenta in the two groups.②HBV DNA copies were measured by Fluorescence Quantitative PCR(FQ-PCR).③The human trophoblast cell line Bewo were co-cultured with HBV DNA positive sera. Immunohistochemistry ABC was conducted to detect distribution of HBsAg in Bewo.④Using semi-quantitative RT-PCR and quantitative Real-time RT-PCR to explore the change of TLR3 mRNA level after exposed to HBV in Bewo,to exploe the change of TLR3 mRNA leve after exposed to the TLR3 agonist polyI:C and siRNA.Measuring HBV DNA copies in culture supernatant after co-culturing with HBV for 24 and 48 hours in the two groups and the control group. RESULTS:①TLR3 mRNA in term placenta showed weaker expression in HBsAg-positive pregnant women than normal pregnant women by both semi-quantitative RT-PCR and quantitative Real-time RT-PCR(t=3.003,P＜0.05;t=4.698,P＜0.05).The positive rate of TLR3 in normal and HBsAg-positive pregnant women was 100%(41/41) and 73.0(85/116) respectively. TLR3 was mostly located in the cytoplasm,a part on the cell membrane and occasionally in the nuceus.TLR3 positive rate gradually increased from the maternal side to the fetal side(trend test P＜0.001) in the placental cell layers.There was significant difference in distribution of TLR3 between placentas of normal and HBsAg-positive pregnant women(Fisher's exat probability test P＜0.05).②There was no relation between TLR3 mRNA levels in term placenta and HBV DNA copies in peripheral blood in HBsAg-positive pregnant women(F=0.147,P＞0.05).There was no relation between TLR3 mRNA levels in term placenta and HBV interuterine infections in newborns(t=0.888,P＞0.05).③Immunohistochemistry ABC and double labeling immunofluorescent histochemistry assay showed TLR3 expression is a protective factor either in placenta HBV infection(OR=0.259, 0.189～0.354) or in HBV interuterine infections in newborns(OR=0.438,0.204～0.942).④HBV DNA were detected in culture supernatant after co-culturing for 24and 48 hours with HBV DNA positive sera.Expression of HBsAg was successfully detected in Bewo cells by Immunohistochemistry ABC.⑤Expression of TLR3 mRNA was was successfully detected in Bewo cells by semi-quantitative RT-PCR and quantitative Real-time RT-PCR.TLR3 mRNA showed stronger expression after exposed to HBV in Bewo cells(t=4.479,P＜0.05).TLR3 mRNA showed stronger expression after exposed to Poly I:C than exposed to siRNA and control group(H=18.984,P＜0.05).After co-cultured with 10~8 copies HBV DNA,the supernatant of Bewo cells culture showed HBV DNA positive in all three groups.The concentrations of HBV DNA in Poly I:C groups was significantly lower than those in other two groups(F=8.531, P＜0.05).But there was no significant difference between siRNA groups and the control group.CONCLUSION:①TLR3 expression in placenta is a protective factor to HBV infection in pregnant women.②TLR3 mRNA level may have no effect on HBV intrauterine infection.But at protein level,TLR3 take the role against HBV infection in placenta and intrauterine in newborns.③The human trophoblast celll line Bewo can express TLR3 consistently.Bewo cells can be infected by HBV through co-culture with it.So the model can be used to study the mechanism of HBV infection in placenta.④Both Poly I:C and HBV stimulation can upregulate TLR3 mRNA in Bewo cells.